My laboratory continues to investigate the role of Sonic hedgehog (Shh) and its downstream signaling components in developing and mature nervous system. THE ROLE OF GLI3 IN EMBRYONIC NEUROGENESIS: Shh signaling pathway is transduced via two major effectors, Gli2 as an activator and Gli3 as a repressor following a posttranslational modification. Since Gli3 null mice have severe neural patterning defects and die at birth, we utilized conditional gene ablation approach with Nestin-Cre mice to address the role of Gli3 in later neural development. First, we found that the generation of intermediate progenitors during the cortical development is greatly disrupted in the absence of Gli3, resulting in thinner neocortex with more pronounced defects in the formation of superficial cortical layers II/III. As a result, the corpus callosum, which is the major projections emanating from layers II/III neurons, is greatly reduced in the postnatal forebrain of the Gli3 conditional mutants. Taken together with in utero electroporation experiments, our results demonstrate the importance of the negative regulator of Shh pathway, Gli3, in generation of intermediate progenitors during embryonic neurogenesis that ultimately impact on proper cortical neuron specification. THE ROLE OF GLI2 OR GLI3 IN ESTABLISHMENT ANDN MAINTENANCE OF ADULT NSCS AND THEIR NICHE: Adult forebrain continuously generate new neurons in two discrete regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus. By the end of embryonic neurogenesis in rodents, Radial Glial Cells (RGCs) become postnatal neural stem cells (NSCs) as well as the ependymal cells. Interestingly, these RGC-like cells expressing NSC markers persist in postnatal forebrains of Gli3 conditional mutants while the ependymal cells lining the wall of the lateral ventricle are greatly mis-shaped and sparsely placed. Further molecular marker analysis reveal that cytoarchitecture of the SVZ is greatly disrupted: NSCs and neuroblasts appear in clusters and protrude directly to the lumen of the lateral ventricle. As a consequence of defects in proper cell type transition and migration, the olfactory bulb is greatly reduced in size. We are currently investigating the molecular mechanism by which Gli3 impacts on the establishment of NSCs. In addition, we plan to dissect out the role of Gli3 specifically in the ependymal cells vs. NSCs in the SVZ by conditionally ablating Gli3 with the FoxJ1-Cre mice (ependymal-specific deletion) or GFAP-Cre mice (NSCs and astrocytes). In order to investigate the role of Gli3 or Gli2 in maintenance of NSCs in postnatal brains, we have begun analysis of conditional mutants with a specific ablation of Gli2 or Gli3 in Shh-responding NSCs using Gli1-CreER mice. GENE EXPRESSION PROFILING OF ADULT NSCS AND THEIR NICHE: We have isolated Shh-responding NSCs based on their expression of Gli1 and GFAP using fluorescence-activate cell sorting (FACS) from two neurogenic regions of the forebrain, SVZ of the lateral ventricle and DG of hippocampus. We compared gene expression profiles obtained from Affymetrix microarray experiments to that from non-neurogenic region, the cerebellum, where Gli1 and GFAP expressing cells are Bergman Glia cells. Initial analysis indicates that the overall profiles of the neurogenic SVZ and hippocampus are significantly different from that of non-neurogenic cerebellum. In brief, we found 66 genes and 125 genes to be differentially expressed between hippocampus and SVZ, respectively, when compared to the cerebellum. Among these distinct genes, 16 genes are expressed in both SVZ and hippocampus. We are currently validating the gene expression profiles using RT-PCR and RNA in situ hybridization. Once we narrow down the list of genes through validation processes, we will investigate the functional significance of thesse genes in NSC biology in vitro and in vivo. In addition, we are comparing the gene expression profiles of NSCs to that of the putative neurogenic niche cells including astrocytes, ependymal and endothelial cells in the SVZ. Since the behavior of NSCs is dictated by the extrinsic cues provided in their niche, we are specifically interested in the signaling molecules such as secreted ligands and their receptors and extracellular cell-cell contact molecules. Using various genetically modified mouse lines and fluorescent reporter lines, we plan to isolate each cell population using FACS and perform the gene expression profiling using Signal Sequence Trapping (SST) method. We have validated the specificity of the cell types labeled in these various mouse lines using immunohistochemistry and will be launching on FACS isolation in near future. Compared to microarray experiments designed to identify specific genes downstream of Shh signaling, this alternative SST provides possible interacting components (either through secretion or cell-cell contact) between NSCs and their putative niches in an unbiased manner. HIPPOCAMPAL NEUROGENESIS: Another neurogenic region in the adult forebrain is the subgranular zone (SGZ) of the dentate gyrun in the hippocampus. New granule neurons are continuously generated and integrated into the existing neural circuits. As in the SVZ of the lateral ventricle, NSCs in the SGZ of the dentate gyrus also respond to Shh signaling. First, we have investigated when the Shh-responding NSCs are established in the hippocampus by profiling the expression patterns of Shh and its response gene, Gli1 starting at E18.5 to neonatal stages using Shh-Cre:GFP and Gli1-nLacZ mice. We found that the Shh-responding (Gli1-expressing) cells start to appear in the DG at the late gestational stage and increase in number in the neonatal stages. However, the location of Shh-responding cells are restricted to the SGZ and are not found in other hippocampal structures. In contrast, Shh-expressing cells first appear in the hilus region of the dentate gyrus and CA3 region of the hippocampus at E18.5 and persist in postnatal stages. Interestingly, the cumulative fate mapping of Shh-lineage (Shh-Cre;Tau-mGFP) reveal that the cells expressing Shh do not turn over in number and send projections to innervate the molecular layer of the dentate gyrus. In contrast, Shh-responding cells expand in number as the NSCs proliferate and generate more granule neurons, which mature over time to innervate CA3 pyramidal neurons. We are in the process of characterizing the molecular nature of the Shh- and Gli1-lineage cells using immunohistochemistry. THE GENETIC LINEAGES OF MIDBRAIN DOPAMINERGIC NEURONS: Dopaminergic (DA) neurons in the ventral midbrain are diverse in their spatial distribution and neural circuits that ultimately results in various physiological functions. We are currently investigating the origin of diversity among midbrain DA neurons by analyzing the contribution of two genetic lineages, Shh and Gli1. Temporal fate mapping results using Shh-CreER and Gli1-CreER mice show that Shh and Gli1-lineage cells contribute to somewhat distinct population of DA neurons along the medial/lateral and anterior/posterior axes. The study will be extended to postnatal mature brain to investigate the neural circuitry established by these two genetic lineages coming from distinct developmental periods. In addition, we have begun gain-of-function and loss-of-function experiments using ROSA26-loxP-STOP-loxP-SmoM2 and SmoFlox mice, respectively, to perturb Shh signaling in distinct spatial and temporal domains to address the requirement of Shh signaling in DA neuron diversity.